Cdc42 and PI(4,5)P2-induced actin assembly in xenopus egg extracts

Andres M. Lebensohn; Le Ma; Hsin-Yi Henry Ho; Marc W. Kirschner

doi:10.1016/S0076-6879(06)06013-7

Cdc42 and PI(4,5)P₂-induced actin assembly in xenopus egg extracts

Andres M. Lebensohn, Le Ma, Hsin-Yi Henry Ho, Marc W. Kirschner

Research output: Contribution to journal › Article

12 Scopus citations

Abstract

Xenopus egg cytoplasmic extracts have been used to study a variety of complex cellular processes. Given their amenability to biochemical manipulation and physiological balance of regulatory proteins, these extracts are an ideal system to dissect signal transduction pathways leading to actin assembly. We have developed methods to study Cdc42 and PI(4,5)P₂-induced actin assembly in Xenopus egg extracts. In this chapter, we describe detailed procedures to prepare Xenopus egg extracts, Cdc42, and PI(4,5)P₂ for use in actin assembly experiments. We also describe a fluorometric pyrene actin assay for quantitative kinetic analysis of actin polymerization and a microscopic rhodamine actin assay for quick measurement of actin rearrangements in extracts. Finally we provide a protocol for immunodepletion of proteins and discuss the use of immunodepletion and rescue experiments for functional analysis of components in the extracts.

Original language	English (US)
Article number	13
Pages (from-to)	156-173
Number of pages	18
Journal	Methods in Enzymology
Volume	406
DOIs	https://doi.org/10.1016/S0076-6879(06)06013-7
State	Published - 2006
Externally published	Yes

ASJC Scopus subject areas

Biochemistry

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Lebensohn, A. M., Ma, L., Ho, H-Y. H., & Kirschner, M. W. (2006). Cdc42 and PI(4,5)P₂-induced actin assembly in xenopus egg extracts. Methods in Enzymology, 406, 156-173. [13]. https://doi.org/10.1016/S0076-6879(06)06013-7

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abstract = "Xenopus egg cytoplasmic extracts have been used to study a variety of complex cellular processes. Given their amenability to biochemical manipulation and physiological balance of regulatory proteins, these extracts are an ideal system to dissect signal transduction pathways leading to actin assembly. We have developed methods to study Cdc42 and PI(4,5)P2-induced actin assembly in Xenopus egg extracts. In this chapter, we describe detailed procedures to prepare Xenopus egg extracts, Cdc42, and PI(4,5)P2 for use in actin assembly experiments. We also describe a fluorometric pyrene actin assay for quantitative kinetic analysis of actin polymerization and a microscopic rhodamine actin assay for quick measurement of actin rearrangements in extracts. Finally we provide a protocol for immunodepletion of proteins and discuss the use of immunodepletion and rescue experiments for functional analysis of components in the extracts.",

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